Zheng Feng-li, Zhao Jia-hui, Zhang Hua-ping
Shanxi Medical University, Taiyuan, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2012 Apr;40(4):327-31.
Genistein could inhibit the development of atherosclerosis. This study explored the role of PI3K/AKT signaling during genistein promoted eNOS activation.
Human umbilical vein endothelial cells (HUVECs) were incubated with ox-LDL (100 mg/L), then treated with genistein (100 nmol/L) for 5, 10, 15, 30 and 60 min. The production of NO was assessed by Griess reaction in cell culture supernatant. The mRNA expression of endothelial nitric oxide synthase (eNOS) was detected by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression of eNOS and phosphorylation eNOS(Ser(1179)) were determined by Western blot. The effect of genistein on phosphorylation eNOS(Ser(1179)) level was also observed in the presence of LY294002 or NSC154020 (PI3K and AKT inhibitors).
The concentration of NO and the expression level of phosphorylation eNOS(Ser(1179)) were significantly increased in ox-LDL + genistein treated cells than ox-LDL treated cells (all P < 0.05), and the peak effects were observed at 15 min, however, eNOS mRNA and non-phosphorylated eNOS protein expression were similar between the two groups (P > 0.05). Furthermore, the expression level of phosphorylation eNOS(Ser(1179)) was significantly lower in PIK3/AKT inhibitors LY294002 and NSC154020 treated cells compared with ox-LDL + genistein treated cells (all P < 0.05).
Genistein could promote the activity of eNOS through increasing phosphorylation eNOS(Ser(1179)) level through PI3K/AKT pathway.
染料木黄酮可抑制动脉粥样硬化的发展。本研究探讨PI3K/AKT信号通路在染料木黄酮促进内皮型一氧化氮合酶（eNOS）激活过程中的作用。
将人脐静脉内皮细胞（HUVECs）与氧化型低密度脂蛋白（ox-LDL，100 mg/L）共同孵育，然后用染料木黄酮（100 nmol/L）处理5、10、15、30和60分钟。通过格里斯反应评估细胞培养上清液中一氧化氮（NO）的生成。采用逆转录-聚合酶链反应（RT-PCR）检测内皮型一氧化氮合酶（eNOS）的mRNA表达。通过蛋白质免疫印迹法测定eNOS和磷酸化eNOS（Ser(1179)）的蛋白表达。在存在LY294002或NSC154020（PI3K和AKT抑制剂）的情况下，观察染料木黄酮对磷酸化eNOS（Ser(1179)）水平的影响。
与ox-LDL处理的细胞相比，ox-LDL +染料木黄酮处理的细胞中NO浓度和磷酸化eNOS（Ser(1179)）的表达水平显著升高（均P < 0.05），在15分钟时观察到峰值效应，然而，两组之间eNOS mRNA和非磷酸化eNOS蛋白表达相似（P > 0.05）。此外，与ox-LDL +染料木黄酮处理的细胞相比，PIK3/AKT抑制剂LY294002和NSC154020处理的细胞中磷酸化eNOS（Ser(1179)）的表达水平显著降低（均P < 0.05）。
染料木黄酮可通过PI3K/AKT途径增加磷酸化eNOS（Ser(1179)）水平，从而促进eNOS的活性。
